Genetic information written in DNA is transcribed into RNA in various combinations, to produce complicated phenotypes of organisms. Contribution of RNA to phenotypes of organisms is known to be dependent on the types and expression levels of the RNA, and extraction of highly pure RNA from various biological materials is important for performing gene expression analysis. For achievement of this object, many methods for extraction of RNA have been developed so far. Examples of methods for isolation of RNA frequently employed include phenol extraction, precipitation from chaotropic salt solutions and adsorption to silica membranes.
Patent Document 1 discloses a solution for RNA extraction comprising 2 to 5 M guanidine and 40 to 60% phenol. RNA extraction had required not less than 2 days of operation using an ultracentrifuge before, but use of this solution enabled efficient extraction of RNA in 3 hours. This method is called the single-step method.
By improvement of the method described in the above Patent Document 1, Patent Document 2 discloses an extraction solution for simultaneous extraction and separation of RNA, DNA and proteins from a sample comprising of these components. More specifically, the literature describes extraction and separation of RNA into an aqueous layer by using a 30 to 50% phenol solution containing 0.5 to 2 M guanidine.
Although the solutions described in Patent Documents 1 and 2 have different compositions, RNA can be extracted by similar operations using the solutions. That is, each solution is used for homogenization of a biological tissue, and a hydrophobic organic solvent such as chloroform is used upon centrifugation of the homogenate to achieve layer separation. Thereafter, the aqueous layer in the uppermost part comprising RNA is recovered. RNA is then precipitated with alcohol and washed in order to extract RNA.
However, RNA isolated using the solutions and the methods described in Patent Document 1 and 2 still shows contamination with (residual) genomic DNA in an amount which can be detected by the reverse transcription-polymerase chain reaction assay (RT-PCR), leading to problems such as loss of quantitativeness of RNA in cases of RT-PCR (Patent Document 3, e.g., paragraph 0005). Therefore, RNA isolated by these methods needs to be further purified for removal of DNA as a contaminant.
A commonly used method for removal of DNA contained as an impurity in an extracted RNA sample is treatment of the RNA sample with deoxyribonuclease (DNase). However, in cases where treatment with DNase is carried out in a liquid layer, it is necessary to perform phenol/chloroform extraction and denaturation of proteins again for removal of DNase after the treatment. Further, in cases where the extraction is performed using a combination of silica membrane columns, the operation of washing the columns needs to be carried out repeatedly. Although contamination with DNA is reduced by this treatment with DNase, such additional labor is required and loss of RNA occurs, resulting in a decreased amount of extracted RNA, which is problematic.
As a method for avoiding contamination of an RNA sample with DNA without performing DNase treatment, Patent Document 3 reports a method using an RNA extraction reagent at a pH of less than 4. However, it is well known that nucleic acid is depurinated and degraded under acidic conditions, and it is therefore difficult to isolate substantially intact RNA. Further, since the solution equilibrium of DNA into the aqueous/organic layer under acidic conditions is biased toward distribution into the organic layer, the effect of suppressing contamination of the aqueous phase with genomic DNA can be expected to some extent by using a reagent for extraction of RNA at a pH of less than 4, but complete suppression of contamination with small DNA fragments having small numbers of bases is impossible.